The susceptibilities of 106 strains of Aeromonas salmonicida to trimethoprim/sulfamethoxazole (SFT) were determined in two laboratories using the Clinical and Laboratory Standards Institute's M42-A disc diffusion protocols. The data generated by the use of discs containing 25 μg SFT (SFT25) allowed the strains to be placed into two groups. Strains in one group (17 strains) generated no inhibition zones and the zones obtained from the other 89 strains were distributed over a wide range but showed no natural division into separate sub-classes. A further investigation performed by one of the participating laboratories, of the susceptibility of 91 of these 106 strains used discs containing 100 μg sulfmethoxazole (SFM100) and 5 μg trimethoprim (TMP5). Application of normalised resistance interpretation to these data allowed the estimation of epidemiological cut-off values for WT strains of ≥ 9 mm for SFM100 and ≥ 21 mm for TMP5. This investigation demonstrated the presence of three distinct phenotypic classes, one containing strains manifesting wild type susceptibility to both agents, another containing strains manifesting non-wild type susceptibility to both and a third containing strains manifesting wild type susceptibility with respect to TMP but non-wild type with respect to SFM. Analysis demonstrated the inability of SFT25 discs to generate data that allowed the separate identification of strains that were fully susceptible to both TMP and SFM from those that were fully susceptible to TMP but were not fully susceptible to SFM. It is recommended that, in investigation of the susceptibility to potentiated sulphonamides of isolates from diseased fish, separate discs, containing the individual components of the mixture, should be employed.

The potential direct health problems posed to marine-farmed salmonids by the biofouling hydroid Ectopleura larynx (Phylum Cnidaria, Class Hydrozoa) and in situ net washing processes to remove the fouling organisms have not yet been addressed. In an attempt to address the possible impacts, the rate of E. larynx growth on aquaculture nets over a net-cleaning cycle was assessed and Atlantic salmon (Salmo salar) smolts were exposed to hydroid-biofouled nets under experimental challenge. After only 1 week of immersion, there was a high settlement of E. larynx on net panels, with the maximum growth observed after 3 week of immersion. For the challenges trials, experimental treatment groups of S. salar were exposed to hydroid net panels or loose hydroid material for 11 hours under controlled conditions. Gills were examined for signs of gross damage and assigned a histopathological gill score. Prior to the experiment, the gills were healthy and did not show signs of damage from any insult. After exposure to E. larynx, focal areas of epithelial sloughing, necrosis and haemorrhage were visible on the gills under histopathology and a maximum gill score of 4 was observed. These results are the first in an investigation of this kind and suggest that E. larynx can damage the gills of S. salar. Further work on this area is vital to develop a better understanding of the pathogenesis of the damage caused by hydroids and their long-term effects on fish health, growth and survival.

This report described the first detections of koi herpesvirus (KHV) in the Republic of
Ireland in imported koi carp. In both cases the KHV suspicions were confirmed by
molecular diagnosis and the infected stocks culled.

Background: Over recent decades jellyfish have caused fish kill events and recurrent gill problems in marine-farmed salmonids. Common jellyfish (Aurelia spp.) are among the most cosmopolitan jellyfish species in the oceans, with populations increasing in many coastal areas. The negative interaction between jellyfish and fish in aquaculture remains a poorly studied area of science. Thus, a recent fish mortality event in Ireland, involving Aurelia aurita, spurred an investigation into the effects of this jellyfish on marine-farmed salmon.
Methodology/Principal Findings: To address the in vivo impact of the common jellyfish (A. aurita) on salmonids, we exposed Atlantic salmon (Salmo salar) smolts to macerated A. aurita for 10 hrs under experimental challenge. Gill tissues of control and experimental treatment groups were scored with a system that rated the damage between 0 and 21 using a range of primary and secondary parameters. Our results revealed that A. aurita rapidly and extensively damaged the gills of S. salar, with the pathogenesis of the disorder progressing even after the jellyfish were removed. After only 2 hrs of exposure, significant multi-focal damage to gill tissues was apparent. The nature and extent of the damage increased up to 48 hrs from the start of the challenge. Although the gills remained extensively damaged at 3 wks from the start of the challenge trial, shortening of the gill lamellae and organisation of the cells indicated an attempt to repair the damage suffered.
Conclusions: Our findings clearly demonstrate that A. aurita can cause severe gill problems in marine-farmed fish. With aquaculture predicted to expand worldwide and evidence suggesting that jellyfish populations are increasing in some areas, this threat to aquaculture is of rising concern as significant losses due to jellyfish could be expected to increase in the future.

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in post-transcriptional gene regulation. Some viruses encode their own miRNAs and these are increasingly being recognized as important modulators of viral and host gene expression. Cyprinid herpesvirus 3 (CyHV-3) is a highly pathogenic agent that causes acute mass mortalities in carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) worldwide. Here, bioinformatic analyses of the CyHV-3 genome suggested the presence of non-conserved precursor miRNA (pre-miRNA) genes. Deep sequencing of small RNA fractions prepared from in vitro CyHV-3 infections led to the identification of potential miRNAs and miRNA–offset RNAs (moRNAs) derived from some bioinformatically predicted pre-miRNAs. DNA microarray hybridization analysis, Northern blotting and stem-loop RT-qPCR were then used to definitively confirm that CyHV-3 expresses two pre-miRNAs during infection in vitro. The evidence also suggested the presence of an additional four high-probability and two putative viral pre-miRNAs. MiRNAs from the two confirmed pre-miRNAs were also detected in gill tissue from CyHV-3-infected carp. We also present evidence that one confirmed miRNA can regulate the expression of a putative CyHV-3-encoded dUTPase. Candidate homologues of some CyHV-3 pre-miRNAs were identified in CyHV-1 and CyHV-2. This is the first report of miRNA and moRNA genes encoded by members of the Alloherpesviridae family, a group distantly related to the Herpesviridae family. The discovery of these novel CyHV-3 genes may help further our understanding of the biology of this economically important virus and their encoded miRNAs may have potential as biomarkers for the diagnosis of latent CyHV-3.

Two laboratories investigated the susceptibility of 106 Aeromonas salmonicida strains (from Denmark, France, Ireland, Norway and Scotland) to erythromycin, gentamicin, oxytetracycline and oxolinic acid using the disc diffusion protocols (M42-A) published by the Clinical and Laboratory Standards Institute. In studies of susceptibility to florfenicol an additional 15 Canadian strains were included. Comparison of the data generated by the two laboratories demonstrated that for each disc both detected a similar pattern of distribution but that there was a significant numerical difference in the zone sizes they recorded. Analysis of the extent of this lateral shift between the data generated in two laboratories indicated that the application of a single laboratory-independent epidemiological cut-off value for each disc could result in disagreement between the laboratories as to whether a strain should be classified as wild-type or non wild-type.
Normalised resistance interpretation was employed to generate epidemiological cut-off values from the data obtained by each laboratory. The use of these laboratory-specific cut-off values resulted in both laboratories achieving complete agreement as to the classification of all strains to all agents.

Background
The doctor fish, Garra rufa, has become increasingly popular as a treatment for skin disorders and for pedicures in recent years. Despite this there is very little information available regarding the welfare of these fish and the range of potential pathogens they may carry. In this study, a group of fish suffering from post-transport mortalities were examined and the isolated pathogens identified.
Findings
Group B Streptococcus agalactiae was isolated from kidney swabs of the fish and found to be resistant to a number of antibiotics. In addition to this, a fish virus belonging to the aquabirnavirus group, serogroup C was isolated for the first time in Ireland. However, no clinical signs of disease typical of bacterial or viral infections were observed in any fish examined.
Conclusions
As no clinical signs of disease attributable to either of the pathogens identified were found it was concluded that the mortalities were most likely due to transport related stress exacerbated by the presence of the pathogens. Further work is required to assess the suitability of current transport strategies and to examine the potential risk associated with the transport of live ornamental fish.

Amoebic gill disease (AGD) is a proliferative gill disease of marine cultured Atlantic salmon Salmo salar, with the free-living protozoan Neoparamoeba perurans being the primary
aetiological agent. The increased incidence of AGD in recent years presents a significant challenge to the Atlantic salmon farming industry in Europe. In this study, a real-time TaqMan® PCR
assay was developed and validated to detect Neoparamoeba perurans on Atlantic salmon gills and further used to monitor disease progression on a marine Atlantic salmon farm in Ireland in conjunction
with gross gill pathology and histopathology. The assay proved specific for N. perurans, with no cross-reactivity with the related species N. pemaquidensis, N. branchiphila or N. aestuarina,
and was capable of detecting 2.68 copies of N. perurans DNA μl−1. Although the parasite was detected throughout the 18 mo period of this study, mortality peaks associated with clinical AGD
were only recorded during the first 12 mo of the marine phase of the production cycle. The initial AGD outbreak resulted in peak mortality in Week 17, which was preceded by PCR detections
from Week 13 onwards. Freshwater treatments were an effective method for controlling the disease, resulting in a reduction in the weekly mortality levels and also a reduction in the number of
PCR-positive fish. In comparison to traditional diagnostic methods, our PCR assay proved to be highly sensitive and a valuable tool to monitor disease progression and, therefore, has the potential
to provide information on the timing and effectiveness of treatments.

During July 1992, an acute clinical outbreak of proliferative kidney disease (PKD) was experienced in two strains (‘Irish’ and ‘Norwegian’) of juvenile (age 0+) Atlantic salmon (Salmo salar L.) held at two adjacent freshwater sites on the River Lee in southern Ireland. Various management strategies (including reduced stocking densities, handling, feeding rates and increased oxygenation), and treatment regimes (involving malachite green and fumagillin DCH) were used to control the disease. A total of 1·3 million juveniles died during the PKD outbreak, representing 61·6% and 54·6% of
the Norwegian stock at the two farms respectively. The Irish stock appeared to be more resistant to the disease and only 15·6% died. The weekly prevalence of PKD fluctuated throughout the summer but seemed to disappear by mid-August. Although PKD was detected again during 1993, no clinical outbreak occurred. In conjunction with the management strategies adopted in 1992, seven consecutive weekly prophylactic bath treatments with malachite green (1·6 ppm for 40 minutes) administered prior to mid-July appeared to control the disease. During August 1993, a ten day course of fumagillin (6 mg/kg bodyweight per day) reduced the prevalence of the PKD parasite in a trial batch of juveniles from 24% to zero. The results of this study demonstrated the effectiveness of various management strategies and treatment regimes in controlling PKD.

Observed emergence of IPNV in farmed Irish salmon is simulated using a model originally developed to analyse the spread of the virus in Scotland [Murray, A.G., 2006a. A model of the spread of infectious pancreatic necrosis virus in Scottish salmon farms 1996–2003. Ecol. Model. 199, 64–72]. IPNV appears to have become established relatively recently in Ireland and the model is altered to explicitly simulate the origin of the spread of the virus. Input to freshwater farms was key to initiation of infection, but modelling suggests that endogenous spread was responsible for much of the subsequent increase in prevalence of IPNV. From the modelling, it is unlikely that direct imports accounted for most IPNV cases. If this is the case, cessation of imports, without a substantial improvement in biosecurity, would be likely to be of only limited effect in controlling IPNV. Marine IPNV prevalence appears to be insensitive to direct interventions in the marine environment (as in the Scottish model). A multi-element control strategy, targeting both endogenous spread and external input of infection and prioritising freshwater sites, but extending to marine sites, would probably now be required to eradicate IPNV from Ireland.

This study investigated the genotypes and sub-groups of infectious pancreatic necrosis virus (IPNV) present in farmed and wild salmonid fish in Ireland. An 1100-bp portion of the VP2 region of segment A from each of 55 IPNV isolates collected over 2003–2007 was amplified by reverse-transcription–polymerase chain reaction and the product directly sequenced. The nucleotide sequences of each isolate were aligned and compared with each other and with the corresponding sequences of a number of reference isolates. All the 55 sequenced isolates belonged to genogroup 5 (Sp serotype) and could be divided into two subgroups. Irish subgroup 1 consisted of isolates from farmed salmon originating from an Irish salmon broodstock. Irish subgroup 2 consisted of isolates from imported farmed stock and all reported clinical outbreaks of IPN were associated with isolates from subgroup 2. Isolates from wild fish were identical to some isolates from subgroup 2, and therefore are believed to have originated from infected farms. These results highlight the importance of import risk analysis for diseases not listed under current legislation.

Infectious pancreatic necrosis is a significant disease of farmed salmonids resulting in direct economic losses due to high mortality and disease-management costs. Significant outbreaks of the disease occurred in farmed Atlantic salmon in Ireland between 2003 and 2007, associated with imported ova and smolts. As the virus was known to occur in the country since the development of aquaculture in the 1980s, this study examined archived samples to determine whether these older isolates were associated with virulent forms. The study showed that two genotypes of IPNV were present in the 1990s, genotype 3 and genotype 5. A more virulent subtype of the virus first appeared in 2003 associated with clinical outbreaks of IPN, and this subtype is now the most prevalent form of IPNV found in the country. The data also indicated that IPNV in Ireland is more closely related to Scottish and continental European isolates than to Norwegian, Chilean and Australasian genogroup 5 isolates.

We describe a disease syndrome that afflicts larval, landlocked Atlantic salmon Salmo salar from Cayuga Lake, one of central New York's Finger Lakes. Mortality associated with the “Cayuga syndrome” is 98–100%. Death usually occurs between 650 and 850 centigrade degreedays after fertilization, approximately 2–4 weeks before yolk resorption is complete. Although there is minor temporal variation in the onset of the Cayuga syndrome in progeny from individual females, all sac fry eventually succumb. Incubation of embryos and sac fry under constant, ambient, or reduced temperature regimens slightly alters the degree-day timing of syndrome onset, but does not improve survival. Based on mortality rate, manifestation of the Cayuga syndrome has not changed in the past 10 years, even though incubation waters of varying chemistry and temperature have been used. Mortality of the negative control stocks used for these studies never exceeded 10% from hatching to first feeding. Findings from reciprocal crossbreeding experiments indicate the problem is associated with ova only. A noninfectious etiology is indicated by the lack of consistently identifiable fish pathogens from syndrome-afflicted sac fry and by the failure to transmit the condition horizontally. Suspect contaminants were eliminated as potential causative factors. Epidemiological studies on the viability of other Finger Lakes stocks indicate that Atlantic salmon from Keuka and Seneca lakes are also afflicted (100% mortality). yet those from Skaneateles Lake are not. The cause of this syndrome appears to be nutritional.

The Pacific oyster, Crassostrea gigas, plays a significant role in the aquaculture industry in Ireland. Episodes of increased mortality in C. gigas have been described in many countries, and in Ireland since 2008. The cause of mortality events in C. gigas spat and larvae is suspected to be multifactorial, with ostreid herpesvirus 1 (OsHV-1, in particular OsHV-1 μvar) considered a necessary, but not sufficient, cause. The objectives of the current study were to describe mortality events that occurred in C. gigas in Ireland during the summer of 2011 and to identify any associated environmental, husbandry and oyster endogenous factors. A prospective cohort study was conducted during 2010–2012, involving 80 study batches, located at 24 sites within 17 bays. All 17 bays had previously tested positive for OsHV-1 μvar. All study farmers were initially surveyed to gather relevant data on each study batch, which was then tracked from placement in the bay to first grading. The outcome of interest was cumulative batch-level mortality (%). Environmental data at high and low mortality sites were compared, and a risk factor analysis, using a multiple linear regression mixed effects model, was conducted. Cumulative batch mortality ranged from 2% to 100% (median = 16%, interquartile range: 10–34%). The final multivariable risk factor model indicated that batches imported from French hatcheries had significantly lower mortalities than non-French hatcheries; sites which tested negative for OsHV-1 μvar during the study had significantly lower mortalities than sites which tested positive and mortalities increased with temperature until a peak was reached. There were several differences between the seed stocks from French and non-French hatcheries, including prior OsHV-1 μvar exposure and ploidy. A range of risk factors relating to farm management were also considered, but were not found significant. The relative importance of prior OsHV-1 μvar infection and ploidy will become clearer with ongoing selection towards OsHV-1 μvar resistant oysters. Work is currently underway in Ireland to investigate these factors further, by tracking seed from various hatchery sources which were put to sea in 2012 under similar husbandry and environmental conditions.

Viral gametocytic hypertrophy (VGH) was detected during an investigation of mortalities in Pacific oysters Crassostrea gigas from 2 separate Irish production sites. The basophilic inclusions were observed in the gonad tissue of oysters sampled in August and October 2007. The oysters involved did not show any macroscopic disease signs. Transmission electron microscopy demonstrated the presence of viral particles in these intranuclear inclusions. The particles were small, non-enveloped, icosahedral and approximately 50 nm in diameter and thus had characteristics similar to the Papillomaviridae and Polyomaviridae families. No host defence reaction was observed. The viral particles described here appear to be similar to those described in C. virginica from the USA and Canada and to those described in C. gigas from Korea and France.

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